Characterization of the activity of KTX-1001, a small molecule inhibitor of multiple myeloma SET domain using surface...

KTX-1001 is a small molecule inhibitor of NSD2 in clinical development for multiple myeloma. It was identified as a potent and selective inhibitor of NSD2 (also known as MMSET) using a high-throughput biochemical assay with LC/MS-MS detection of SAH production as the endpoint. Subsequent evaluation of the binding of KTX-1001 to its target was conducted using surface plasmon resonance to quantify on-rate, off-rate, and equilibrium dissociation constant utilizing the SET domain as the immobilized target. In this format, no saturable or specific binding could be observed, despite the potent activity in the biochemical assay using full length NSD2 and radiolabeled SAM. To interrogate the discordance between potent activity and the lack of detectable binding, a series of experiments were designed in which KTX-1001 with a biotin-PEG tether (KTX-1001-3) was immobilized to the chip, with target (NSD2 SET domain) in-flow, with nucleosomes and with and without cofactor. These experiments demonstrated that KTX-1001-3 bound to the SET domain in a specific and saturable manner, with an affinity comparable to the IC₅₀ determined in the enzymatic assay. Further, these studies confirmed unique binding properties of KTX-1001 in the presence of nucleosomes, cofactor, and in combination. These data identify the utility of surface plasmon resonance in a "reverse" format, where immobilization of KTX-1001 allowed for the interrogation of binding to a protein target that may be challenging in the conventional SPR format. Collectively, this analysis demonstrates the specific potent biochemical activity of KTX-1001 against MMSET and supports the ongoing evaluation of KTX-1001 in the clinic.